The proposed work will develop a sequence-based strain differentiation method for Mycobacterium tuberculosis (Mtb). The PI will first locate a chromosomal segment with substantial sequence variability among Mtb strains. The segment chosen will be flanked by conserved sequences, which will serve as PCR primer sites. This will allow the variable segment to be amplified and sequenced rapidly in many Mtb strains. The PI has already identified a segment which is moderately variable among M. avium complex strains. However, this segment shows no sequence variability in Mtb. The Research Plan includes several alternate methods for the location of a variable chromosomal segment in Mtb (major polymorphic tandem repeat sequencing, restriction site polymorphism mapping, and representational difference analysis). The strain differentiation method will be applied to a well-characterized CDC collection of Mtb strains from New York City. About 100 of the strains will be sequenced and grouped into a statistically-validated phylogeny. In a parallel analysis, the PI identified a distinct phylogenetic group of M. avium complex strains associated with disease in AIDS patients. The phylogenetic placement of Mtb strains from patients with and without HIV infection will be compared. Similar comparisons will be made according to other clinical factors (IV drug abuse, ethnicity, site of infection, primary vs. secondary isolate, and antibiotic susceptibility). The association of phylogenetic groups of Mtb with specific patterns of disease would provide a framework for studies of virulence and pathophysiology. In a separate analysis, a phylogeny of Mtb isolates from various parts of the world will be constructed. The method developed will also be applied to rapid diagnosis of multidrug- resistant Mtb (MDR-TB). Oligonucleotide probe sites specific for individual strains will be identified from the sequence data. A PCR/hybridization assay will be developed to rapidly detect particular outbreak-associated MDR-TB strains directly in clinical specimens. The assay will be validated in an ongoing outbreak. Rapid diagnosis of MDR-TB will allow early appropriate antibiotic choice.